Neurodegenerative diseases, such as Alzheimer's, Parkinson's, Huntington's disease (HD) and ALS are a major health risk in the US affecting millions of people. All of these diseases are fata and no therapeutic is available that blocks the progression of any neurodegenerative disease. One of the reasons that no effective therapies exist is our lack of understanding of the specific molecular mechanisms that cause neurodegeneration and disease progression. Our goal is to employ novel technologies to begin to better understand the molecular basis of neurodegeneration. To do this, we will collaborate with Dr. Steven Finkbeiner of the Gladstone Institute of Neurodegenerative Diseases in studies employing induced Pluripotent Stem Cells (iPSC) from patients with HD and ALS. Dr. Finkbeiner, as part of the HD iPSC Consortium, recently described the development of HD patient derived iPSCs that were differentiated to striatal neurons (i-neurons) the cells most affected in the disease. The i-neurons expressed disease phenotypes including shorter survival times compared to i-neurons from healthy individuals. Using a novel technology, nanoGenomics, we propose begin to identify cellular pathways which are disrupted in HD neurons that may be linked to neurodegeneration. To do this, we will conduct whole transcriptome analysis (WTA) in individual HD patient and control i-neurons. This single cell analysis will allow us to directly identify the progression of genetic ad molecular pathway changes at different stages of neurodegeneration of i-neurons. The basis of our nanoGenomics technology is a nanopipette platform that we invented that allows for voltage controlled aspiration of single i-neuron content. We combine this single cell analysis with a unique strategy for RNA sequencing of individual cells that requires less than 500 pg of total RNA which we have already validated to measure gene expression profiles in single breast cancer cells. The WTA will reveal clusters of genes that are differentially expressed in the control and HD i-neurons. Using informatics, we will relate this cluster analysis to cellular pathways dysregulated in HD i- neurons. The WTA results will be verified by qPCR. Using a novel nanopipette-based nanosyringe technology, we will extract small samples (15 femtomoles) of cellular content over time. This procedure does not affect cell viability and will allow us to identify temporal changes in these cellular pathways in individuals HD i-neurons to link molecular changes to neurodegeneration. We will expand the utility of nanoGenomics to other neurodegenerative diseases such as ALS. Dr. Finkbeiner and associates recently reported the development of iPSC-derived motor neurons (i-MN) from an ALS patient with a mutation in TDP43 which exhibited disease phenotypes. Comparative analysis of the nanoGenomics of ALS with HD will provide insights into common molecular disruptions that cause neurodegeneration as well as mechanisms unique to each disease.